The serotonin 5-HT(2A) receptor (5-HT(2A)R) and dopamine D(2) receptor (D(2)R) are high-affinity G protein-coupled receptor targets for two different classes of antipsychotic drugs used to treat schizophrenia. Interestingly, the antipsychotic effects are not based on the regulation of same signaling mediators since activation of the 5-HT(2A)R and of the D(2)R regulate G(q/11) protein and G(i/o) protein, respectively. Here we use radioligand binding and second messenger production assays to provide evidence for a functional crosstalk between 5-HT(2A)R and D(2)R in brain and in HEK293 cells. D(2)R activation increases the hallucinogenic agonist affinity for 5-HT(2A)R and decreases the 5-HT(2A)R induced inositol phosphate production. In vivo, 5-HT(2A)R expression is necessary for the full effects of D(2)R antagonist on MK-801-induced locomotor activity. Co-immunoprecipitation studies show that the two receptors can physically interact in HEK293 cells and raise the possibility that a receptor heterocomplex mediates the crosstalk observed. The existence of this 5-HT(2A)R-D(2)R heteromer and crosstalk may have implications for diseases involving alterations of serotonin and dopamine systems and for the development of new classes of therapeutic drugs.

Heterotrimeric G proteins are molecular switches that relay information intracellularly in response to various extracellular signals. How ligand-activated G protein-coupled receptors act at a distance to exert exchange activity on the Galpha nucleotide binding pocket is poorly understood. Here we describe the synergistic action of two peptides: one from the third intracellular loop of the D2 dopamine receptor (D2N), and a second, Galpha.GDP-binding peptide (KB-752) that mimics the proposed role of Gbetagamma in receptor-promoted nucleotide exchange. The structure of both peptides in complex with Galpha(i1) suggests that conformational changes in the beta3/alpha2 loop and beta6 strand act in concert for efficient nucleotide exchange. Two key residues in the alpha4 helix were found to define a receptor/Galpha(i) coupling specificity determinant.

Binding of dopamine receptor ligands to human D2 and D3 receptors was characterized in Chinese hamster ovary (CHO) cells using the dopamine D2 receptor antagonist [125I] iodosulpiride. Only limited binding selectivity was observed for known dopamine D2 receptor antagonists from a variety of chemical classes, which included haloperidol, chlorpromazine, sulpiride, pimozide and cis flupenthixol. The most selective compound from this group were (+)butaclamol and domperidone which showed 5-fold D3 selectivity. A number of high affinity dopamine receptor agonists, including apomorphine and bromocriptine, also failed to demonstrate selectivity. In contrast, the natural ligand dopamine and the efficacious synthetic agonists quinpirole, (+)4-propyl-9-hydroxynapthoxazine (PHNO), 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (6,7-ADTN), 7-OH DPAT and N-0434 showed marked apparent human dopamine D3 (hD3) receptor selectivity. In the aminotetralin series, this selectivity was observed preferentially with analogs of the 6,7-rotamer compared with compounds from the 5,6-rotamer series. Functional coupling of the hD3 receptor was investigated in a number of cell lines in which the hD3 receptor was stably expressed, including CHO cells, the neuroblastoma-glioma hybrid cell line NG108-15 and a rat 1 fibroblast cell line. There was no evidence of functional coupling of the hD3 receptor to adenylate cyclase, arachidonic acid release, phospholipase C activation, K+ currents or calcium mobilization in any of the cell lines examined. Furthermore, guanine nucleotides failed to inhibit the binding of [3H] N-0437 to hD3 receptors in any of the three cell lines. There may be a number of explanations for these results. These cell lines may not have the appropriate G-protein or secondary messenger systems that are coupled to the hD3 receptor in situ. Alternatively, this receptor may couple by a mechanism that is as yet undefined. The finding that a wide range of structurally diverse human dopamine D2 (hD2) receptor agonists have an apparent hD3 selectivity may imply that the hD3 receptor exists predominantly in a high affinity state.

Cloned human dopamine D2 receptor cDNA was isolated from a pituitary cDNA library and found to encode an additional 29 amino acid residues in the predicted intracellular domain between transmembrane regions 5 and 6 relative to a previously described rat brain D2 receptor. Results from polymerase chain reactions as well as in situ hybridization revealed that mRNA encoding both receptor forms is present in pituitary and brain of both rat and man. The larger form was predominant in these tissues and, as shown in the rat, expressed by dopaminergic and dopaminoceptive neurons. Analysis of the human gene showed that the additional peptide sequence is encoded by a separate exon. Hence, the two receptor forms are generated by differential splicing possibly to permit coupling to different G proteins. Both receptors expressed in cultured mammalian cells bind [3H]spiperone with high affinity and inhibit adenylyl cyclase, as expected of the D2 receptor subtype.

Interacting selectively and non-covalently with a drug, any naturally occurring or synthetic substance, other than a nutrient, that, when administered or applied to an organism, affects the structure or functioning of the organism; in particular, any such substance used in the diagnosis, prevention, or treatment of disease.

Although the biological basis of schizophrenia is not known, possible causes include genetic defects, viruses, amines, brain structure and metabolism, neuroreceptors, and G proteins. The hypothesis of dopamine overactivity in schizophrenia is based on the fact that neuroleptics block dopamine D2 receptors in direct relation to their clinical antipsychotic potencies. Moreover, dopamine D2 or D2-like receptors are elevated in postmortem schizophrenia brain tissue. This elevation, however, is only found in vivo using [11C]methylspiperone but not [11C]raclopride. The dopamine D4 receptor gene has not yet been excluded in schizophrenia because the 21 gene variants of D4 have not yet been tested. Because the link between D1 and D2 receptors is reduced in schizophrenia tissue, we tested whether one component of this link was sensitive to guanine nucleotide. We report here that the binding of [3H]raclopride to D2 receptors in schizophrenia was not sensitive to guanine nucleotide. This finding permitted analysis of data on the binding of [3H]emonapride to the D2, D3 and D4 receptors. We conclude that the combined density of D2 and D3 receptors (labelled by [3H]raclopride) is increased by only 10% in schizophrenia brain, as found by Farde et al., but that it is the density of dopamine D4 receptors which is sixfold elevated in schizophrenia. These findings resolve the apparent discrepancy, mentioned above, wherein the density of [11C]methylspiperone-labelled sites (D2, D3 and D4), but not that of [11C]raclopride-labelled sites (D2 and D3), was found elevated in the schizophrenia striatum.

G-protein-coupled receptors are generally thought to be organized as dimers; whether they form higher order oligomers is a topic of much controversy. We combined bioluminescence/fluorescence complementation and energy transfer to demonstrate that at least four dopamine D2 receptors are located in close molecular proximity in living mammalian cells, consistent with their organization as higher order oligomers at the plasma membrane. This implies the existence of multiple receptor interfaces. In addition to the symmetrical interface in the fourth transmembrane segment (TM4) we identified previously by cysteine (Cys) crosslinking, we now show that a patch of residues at the extracellular end of TM1 forms a second symmetrical interface. Crosslinking of D2 receptor with Cys substituted simultaneously into both TM1 and TM4 led to higher order species, consistent with our novel biophysical results. Remarkably, the rate and extent of crosslinking at both interfaces were unaltered over a 100-fold range of receptor expression. Thus, at physiological levels of expression, the receptor is organized in the plasma membrane into a higher order oligomeric structure.

A large number of studies have demonstrated co-purification or co-immunoprecipitation of receptors with G proteins. We have begun to look for the presence of effector molecules in these receptor complexes. Co-expression of different channel and receptor permutations in COS-7 and HEK 293 cells in combination with co-immunoprecipitation experiments established that the dopamine D(2) and D(4), and beta(2)-adrenergic receptors (beta(2)-AR) form stable complexes with Kir3 channels. The D(4)/Kir3 and D(2) receptor/Kir3 interaction does not occur when the channel and receptor are expressed separately and mixed prior to immunoprecipitation, indicating that the interaction is not an artifact of the experimental protocol and reflects a biosynthetic event. The observed complexes are stable in that they are not disrupted by receptor activation or modulation of G protein alpha subunit function. However, using a peptide that binds Gbetagamma (betaARKct), we show that Gbetagamma is critical for dopamine receptor-Kir3 complex formation, but not for maintenance of the complex. We also provide evidence that Kir3 channels and another effector, adenylyl cyclase, are stably associated with the beta(2)-adrenergic receptor and can be co-immunoprecipitated by anti-receptor antibodies. Using bioluminescence resonance energy transfer, we have shown that in living cells under physiological conditions, beta(2)AR interacts directly with Kir3.1/3.4 and Kir3.1/3.2c heterotetramers as well as with adenylyl cyclase. All of these interactions are stable in the presence of receptor agonists, suggesting that these signaling complexes persist during signal transduction. In addition, we provide evidence that the receptor-effector complexes are also found in vivo. The observation that several G protein-coupled receptors form stable complexes with their effectors suggests that this arrangement might be a general feature of G protein-coupled signal transduction.

The Ca(2+)-binding protein calmodulin (CaM) has been shown to bind directly to cytoplasmic domains of some G protein-coupled receptors, including the dopamine D(2) receptor. CaM binds to the N-terminal portion of the long third intracellular loop of the D(2) receptor, within an Arg-rich epitope that is also involved in the binding to G(i/o) proteins and to the adenosine A(2A) receptor, with the formation of A(2A)-D(2) receptor heteromers. In the present work, by using proteomics and bioluminescence resonance energy transfer (BRET) techniques, we provide evidence for the binding of CaM to the A(2A) receptor. By using BRET and sequential resonance energy transfer techniques, evidence was obtained for CaM-A(2A)-D(2) receptor oligomerization. BRET competition experiments indicated that, in the A(2A)-D(2) receptor heteromer, CaM binds preferentially to a proximal C terminus epitope of the A(2A) receptor. Furthermore, Ca(2+) was found to induce conformational changes in the CaM-A(2A)-D(2) receptor oligomer and to selectively modulate A(2A) and D(2) receptor-mediated MAPK signaling in the A(2A)-D(2) receptor heteromer. These results may have implications for basal ganglia disorders, since A(2A)-D(2) receptor heteromers are being considered as a target for anti-parkinsonian agents.

Altered synaptic dopamine levels have been implicated in several neurological/neuropsychiatric disorders, including drug addiction and schizophrenia. However, it is unclear what precipitates these changes in synaptic dopamine levels. One of the key presynaptic components involved in regulating dopaminergic tone is the dopamine transporter (DAT). Here, we report that the DAT is also regulated by the dopamine D2 receptor through a direct protein-protein interaction involving the DAT amino-terminus and the third intracellular loop of the D2 receptor. This physical coupling facilitates the recruitment of intracellular DAT to the plasma membrane and leads to enhanced dopamine reuptake. Moreover, mice injected with peptides that disrupt D2-DAT interaction exhibit decreased synaptosomal dopamine uptake and significantly increased locomotor activity, reminiscent of DAT knockout mice. Our data highlight a novel mechanism through which neurotransmitter receptors can functionally modulate neurotransmitter transporters, an interaction that can affect the synaptic neurotransmitter levels in the brain.

A large number of studies have demonstrated co-purification or co-immunoprecipitation of receptors with G proteins. We have begun to look for the presence of effector molecules in these receptor complexes. Co-expression of different channel and receptor permutations in COS-7 and HEK 293 cells in combination with co-immunoprecipitation experiments established that the dopamine D(2) and D(4), and beta(2)-adrenergic receptors (beta(2)-AR) form stable complexes with Kir3 channels. The D(4)/Kir3 and D(2) receptor/Kir3 interaction does not occur when the channel and receptor are expressed separately and mixed prior to immunoprecipitation, indicating that the interaction is not an artifact of the experimental protocol and reflects a biosynthetic event. The observed complexes are stable in that they are not disrupted by receptor activation or modulation of G protein alpha subunit function. However, using a peptide that binds Gbetagamma (betaARKct), we show that Gbetagamma is critical for dopamine receptor-Kir3 complex formation, but not for maintenance of the complex. We also provide evidence that Kir3 channels and another effector, adenylyl cyclase, are stably associated with the beta(2)-adrenergic receptor and can be co-immunoprecipitated by anti-receptor antibodies. Using bioluminescence resonance energy transfer, we have shown that in living cells under physiological conditions, beta(2)AR interacts directly with Kir3.1/3.4 and Kir3.1/3.2c heterotetramers as well as with adenylyl cyclase. All of these interactions are stable in the presence of receptor agonists, suggesting that these signaling complexes persist during signal transduction. In addition, we provide evidence that the receptor-effector complexes are also found in vivo. The observation that several G protein-coupled receptors form stable complexes with their effectors suggests that this arrangement might be a general feature of G protein-coupled signal transduction.

Proteins that bind to G protein-coupled receptors have recently been identified as regulators of receptor anchoring and signaling. In this study, actin-binding protein 280 (ABP-280), a widely expressed cytoskeleton-associated protein that plays an important role in regulating cell morphology and motility, was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. The specificity of this interaction was originally identified in a yeast two-hybrid screen and confirmed by protein binding. The functional significance of the D(2) receptor-ABP-280 association was evaluated in human melanoma cells lacking ABP-280. D(2) receptor agonists were less potent in inhibiting forskolin-stimulated cAMP production in these cells. Maximal inhibitory responses of D(2) receptor activation were also reduced. Further yeast two-hybrid experiments showed that ABP-280 association is critically dependent on the carboxyl domain of the D(2) receptor third cytoplasmic loop, where there is a potential serine phosphorylation site (S358). Serine 358 was replaced with aspartic acid to mimic the effects of receptor phosphorylation. This mutant (D(2)S358D) displayed compromised binding to ABP-280 and coupling to adenylate cyclase. PKC activation also generated D(2) receptor signaling attenuation, but only in ABP-containing cells, suggesting a PKC regulatory role in D(2)-ABP association. A mechanism for these results may be derived from a role of ABP-280 in the clustering of D(2) receptors, as determined by immunocytochemical analysis in ABP-deficient and replete cells. Our results suggest a new molecular mechanism of modulating D(2) receptor signaling by cytoskeletal protein interaction.

The progression of the adenohypophysis over time from its initial formation until its mature state. The adenohypophysis is the anterior part of the pituitary. It secretes a variety of hormones and its function is regulated by the hypothalamus.

The series of molecular signals generated as a consequence of a dopamine receptor binding to its physiological ligand, where the pathway proceeds with inhibition of adenylyl cyclase and a subsequent decrease in the concentration of cyclic AMP (cAMP).

The human dopamine D2L (long form) and D2S (short form) receptors were expressed separately in mouse Ltk- fibroblast cells to investigate whether there is a difference in transmembrane signaling of these D2 receptors. Both receptors induced two signals, a phosphatidylinositol-linked mobilization of intracellular calcium and an inhibition of cyclic adenosine 3'-5' monophosphate (cAMP) accumulation, each with similar response magnitudes and identical pharmacology. Both calcium and cAMP signals were sensitive to pretreatment with pertussis toxin (PTX), indicating mediation by coupling to Gi/Go proteins. However, the two forms of D2 receptor were distinguished by acute prior activation of protein kinase C (PKC) with 12-O-tetradecanoyl 4 beta-phorbol 13-acetate (TPA): TPA blocked the D2S-mediated increase in cytosolic free calcium concentration ([Ca2+]i) in a concentration-dependent manner (between 10 nM and 1 microM), whereas the D2L receptor-induced increase in [Ca2+]i was resistant to TPA and was only partially (60%) inhibited by 100 microM TPA. By contrast, TPA did not alter the inhibition of cAMP accumulation induced by activation of either D2S or D2L receptors. We conclude that, in the L cell system, prior activation of PKC differentially modulates the transmembrane signaling of the D2L and D2S receptors, preferentially inhibiting the D2S receptor-mediated calcium signal but not altering the dopamine-induced inhibitory cAMP signal of either receptor subtype.

Cloned human dopamine D2 receptor cDNA was isolated from a pituitary cDNA library and found to encode an additional 29 amino acid residues in the predicted intracellular domain between transmembrane regions 5 and 6 relative to a previously described rat brain D2 receptor. Results from polymerase chain reactions as well as in situ hybridization revealed that mRNA encoding both receptor forms is present in pituitary and brain of both rat and man. The larger form was predominant in these tissues and, as shown in the rat, expressed by dopaminergic and dopaminoceptive neurons. Analysis of the human gene showed that the additional peptide sequence is encoded by a separate exon. Hence, the two receptor forms are generated by differential splicing possibly to permit coupling to different G proteins. Both receptors expressed in cultured mammalian cells bind [3H]spiperone with high affinity and inhibit adenylyl cyclase, as expected of the D2 receptor subtype.

Binding of dopamine receptor ligands to human D2 and D3 receptors was characterized in Chinese hamster ovary (CHO) cells using the dopamine D2 receptor antagonist [125I] iodosulpiride. Only limited binding selectivity was observed for known dopamine D2 receptor antagonists from a variety of chemical classes, which included haloperidol, chlorpromazine, sulpiride, pimozide and cis flupenthixol. The most selective compound from this group were (+)butaclamol and domperidone which showed 5-fold D3 selectivity. A number of high affinity dopamine receptor agonists, including apomorphine and bromocriptine, also failed to demonstrate selectivity. In contrast, the natural ligand dopamine and the efficacious synthetic agonists quinpirole, (+)4-propyl-9-hydroxynapthoxazine (PHNO), 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (6,7-ADTN), 7-OH DPAT and N-0434 showed marked apparent human dopamine D3 (hD3) receptor selectivity. In the aminotetralin series, this selectivity was observed preferentially with analogs of the 6,7-rotamer compared with compounds from the 5,6-rotamer series. Functional coupling of the hD3 receptor was investigated in a number of cell lines in which the hD3 receptor was stably expressed, including CHO cells, the neuroblastoma-glioma hybrid cell line NG108-15 and a rat 1 fibroblast cell line. There was no evidence of functional coupling of the hD3 receptor to adenylate cyclase, arachidonic acid release, phospholipase C activation, K+ currents or calcium mobilization in any of the cell lines examined. Furthermore, guanine nucleotides failed to inhibit the binding of [3H] N-0437 to hD3 receptors in any of the three cell lines. There may be a number of explanations for these results. These cell lines may not have the appropriate G-protein or secondary messenger systems that are coupled to the hD3 receptor in situ. Alternatively, this receptor may couple by a mechanism that is as yet undefined. The finding that a wide range of structurally diverse human dopamine D2 (hD2) receptor agonists have an apparent hD3 selectivity may imply that the hD3 receptor exists predominantly in a high affinity state.

De novo generation of a long process of a neuron, that carries efferent (outgoing) action potentials from the cell body towards target cells. Refers to the morphogenesis or creation of shape or form of the developing axon.

1. The objective of this study was to characterize the pharmacology of calcium currents in GH4C1 pituitary cells and determine whether activation of heterologously expressed human dopamine receptors can regulate their function. Human D2(short), D3 and D4.2 receptor cDNA's were separately transfected into GH4C1 cells and whole cell calcium currents were recorded by use of nystatin-perforated patch clamp techniques. 2. High-threshold calcium currents were antagonized in a biphasic manner by the dihydropyridine, nisoldipine. The half-maximally effective concentration for each site was 0.2 nM (pIC50 = 9.78 +/- 0.21, n = 4) and 339 nM (pIC50 = 6.47 +/- 0.12, n = 4). The component of current inhibited by 10 nM nisoldipine was also blocked by omega-conotoxin GVIA (30 +/- 9% at 30 nM, n = 6) or by omega-agatoxin IVA (34 +/- 7% at 100 nM, n = 4). 3. Activation of either D2 or D4 receptors by dopamine (10 microM) or quinpirole (0.1 to 10 microM) reduced the peak calcium current by ca. 20% in the majority of cells studied. No inhibition was observed in control or D3 transfected GH4C1 cell lines. 4. The mobilisation of intracellular calcium by thyrotropin releasing hormone in hD4-GH4C1 cells was also studied using Fura-2 AM microspectrofluorimetry. Thyrotropin releasing hormone caused a concentration-dependent increase in calcium mobilisation with an EC50 of 7 nM. D4 receptor activation had no effect upon either basal or hormone-induced [Ca2+]i transients. 5. These results demonstrate that GH4C1 pituitary cells have at least two types of dihydropyridine sensitive high-threshold calcium currents and that like D2 receptors, human D4 receptors can also regulate calcium channel function.

Binding of dopamine receptor ligands to human D2 and D3 receptors was characterized in Chinese hamster ovary (CHO) cells using the dopamine D2 receptor antagonist [125I] iodosulpiride. Only limited binding selectivity was observed for known dopamine D2 receptor antagonists from a variety of chemical classes, which included haloperidol, chlorpromazine, sulpiride, pimozide and cis flupenthixol. The most selective compound from this group were (+)butaclamol and domperidone which showed 5-fold D3 selectivity. A number of high affinity dopamine receptor agonists, including apomorphine and bromocriptine, also failed to demonstrate selectivity. In contrast, the natural ligand dopamine and the efficacious synthetic agonists quinpirole, (+)4-propyl-9-hydroxynapthoxazine (PHNO), 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (6,7-ADTN), 7-OH DPAT and N-0434 showed marked apparent human dopamine D3 (hD3) receptor selectivity. In the aminotetralin series, this selectivity was observed preferentially with analogs of the 6,7-rotamer compared with compounds from the 5,6-rotamer series. Functional coupling of the hD3 receptor was investigated in a number of cell lines in which the hD3 receptor was stably expressed, including CHO cells, the neuroblastoma-glioma hybrid cell line NG108-15 and a rat 1 fibroblast cell line. There was no evidence of functional coupling of the hD3 receptor to adenylate cyclase, arachidonic acid release, phospholipase C activation, K+ currents or calcium mobilization in any of the cell lines examined. Furthermore, guanine nucleotides failed to inhibit the binding of [3H] N-0437 to hD3 receptors in any of the three cell lines. There may be a number of explanations for these results. These cell lines may not have the appropriate G-protein or secondary messenger systems that are coupled to the hD3 receptor in situ. Alternatively, this receptor may couple by a mechanism that is as yet undefined. The finding that a wide range of structurally diverse human dopamine D2 (hD2) receptor agonists have an apparent hD3 selectivity may imply that the hD3 receptor exists predominantly in a high affinity state.

The process in which a signal is passed on to downstream components within the cell, which become activated themselves to further propagate the signal and finally trigger a change in the function or state of the cell.

The human dopamine D2L (long form) and D2S (short form) receptors were expressed separately in mouse Ltk- fibroblast cells to investigate whether there is a difference in transmembrane signaling of these D2 receptors. Both receptors induced two signals, a phosphatidylinositol-linked mobilization of intracellular calcium and an inhibition of cyclic adenosine 3'-5' monophosphate (cAMP) accumulation, each with similar response magnitudes and identical pharmacology. Both calcium and cAMP signals were sensitive to pretreatment with pertussis toxin (PTX), indicating mediation by coupling to Gi/Go proteins. However, the two forms of D2 receptor were distinguished by acute prior activation of protein kinase C (PKC) with 12-O-tetradecanoyl 4 beta-phorbol 13-acetate (TPA): TPA blocked the D2S-mediated increase in cytosolic free calcium concentration ([Ca2+]i) in a concentration-dependent manner (between 10 nM and 1 microM), whereas the D2L receptor-induced increase in [Ca2+]i was resistant to TPA and was only partially (60%) inhibited by 100 microM TPA. By contrast, TPA did not alter the inhibition of cAMP accumulation induced by activation of either D2S or D2L receptors. We conclude that, in the L cell system, prior activation of PKC differentially modulates the transmembrane signaling of the D2L and D2S receptors, preferentially inhibiting the D2S receptor-mediated calcium signal but not altering the dopamine-induced inhibitory cAMP signal of either receptor subtype.

The specific movement from place to place of an organism in response to external or internal stimuli. Locomotion of a whole organism in a manner dependent upon some combination of that organism's internal state and external conditions.

The memory process that deals with the storage, retrieval and modification of information a long time (typically weeks, months or years) after receiving that information. This type of memory is typically dependent on gene transcription regulated by second messenger activation.

Cloned human dopamine D2 receptor cDNA was isolated from a pituitary cDNA library and found to encode an additional 29 amino acid residues in the predicted intracellular domain between transmembrane regions 5 and 6 relative to a previously described rat brain D2 receptor. Results from polymerase chain reactions as well as in situ hybridization revealed that mRNA encoding both receptor forms is present in pituitary and brain of both rat and man. The larger form was predominant in these tissues and, as shown in the rat, expressed by dopaminergic and dopaminoceptive neurons. Analysis of the human gene showed that the additional peptide sequence is encoded by a separate exon. Hence, the two receptor forms are generated by differential splicing possibly to permit coupling to different G proteins. Both receptors expressed in cultured mammalian cells bind [3H]spiperone with high affinity and inhibit adenylyl cyclase, as expected of the D2 receptor subtype.

Any process that stops, prevents, or reduces the frequency, rate or extent of dopamine receptor protein signaling pathway activity. A dopamine receptor signaling pathway is the series of molecular signals generated as a consequence of a dopamine receptor binding to one of its physiological ligands.

OBJECTIVE: von Willebrand factor (VWF) is acutely released from endothelial cells in response to numerous calcium-raising agents (e.g. thrombin, histamine) and cAMP-raising agents (e.g. epinephrine, adenosine, vasopressin). In contrast, very few inhibitors of endothelial VWF secretion have been described. The neurotransmitter dopamine is a modulator of exocytosis in several endocrine cells, and is possibly involved in the regulation of several endothelial cell functions. We therefore investigated the effect of dopamine on endothelial VWF secretion. RESULTS: Dopamine, D2/D3- and D4-specific agonists inhibited histamine- but not thrombin-induced VWF secretion. Expression of dopamine D2, D3 and D4 receptors was demonstrated by reverse transcription polymerase chain reaction (RT-PCR) in both human aortic (HAEC) and umbilical vein (HUVEC) endothelial cells. D2-D4 agonists did not inhibit histamine-induced rise in [Ca(2+)](i): they inhibited histamine-induced secretion even in the absence of extracellular calcium. Thus, the dopamine effects are not mediated by [Ca(2+)](i)-dependent signalling. D2/D3- and D4-specific agonists inhibited neither the rise in cAMP nor VWF secretion in response to epinephrine and adenosine, arguing against an effect on cAMP-mediated signalling. D1 and D5 receptors were not detected in HAEC or HUVEC by RT-PCR, and the D1/D5-specific agonist SKF 38 393 failed to modulate VWF secretion, arguing against a role for these receptors in endothelial exocytosis. CONCLUSIONS: Dopamine inhibits histamine-induced endothelial exocytosis by activating D2-D4 receptor, via a mechanism distinct from [Ca(2+)](i)-or cAMP-mediated signaling. In contrast, D1 and D5 receptors are not functionally expressed in cultured endothelial cells. Dopamine agonists may be useful as inhibitors of endothelial activation in inflammation and cardiovascular disease.

Any process that stops, prevents, or reduces the frequency, rate or extent of glutamatergic synaptic transmission, the process of communication from a neuron to another neuron across a synapse using the neurotransmitter glutamate.

1. The objective of this study was to characterize the pharmacology of calcium currents in GH4C1 pituitary cells and determine whether activation of heterologously expressed human dopamine receptors can regulate their function. Human D2(short), D3 and D4.2 receptor cDNA's were separately transfected into GH4C1 cells and whole cell calcium currents were recorded by use of nystatin-perforated patch clamp techniques. 2. High-threshold calcium currents were antagonized in a biphasic manner by the dihydropyridine, nisoldipine. The half-maximally effective concentration for each site was 0.2 nM (pIC50 = 9.78 +/- 0.21, n = 4) and 339 nM (pIC50 = 6.47 +/- 0.12, n = 4). The component of current inhibited by 10 nM nisoldipine was also blocked by omega-conotoxin GVIA (30 +/- 9% at 30 nM, n = 6) or by omega-agatoxin IVA (34 +/- 7% at 100 nM, n = 4). 3. Activation of either D2 or D4 receptors by dopamine (10 microM) or quinpirole (0.1 to 10 microM) reduced the peak calcium current by ca. 20% in the majority of cells studied. No inhibition was observed in control or D3 transfected GH4C1 cell lines. 4. The mobilisation of intracellular calcium by thyrotropin releasing hormone in hD4-GH4C1 cells was also studied using Fura-2 AM microspectrofluorimetry. Thyrotropin releasing hormone caused a concentration-dependent increase in calcium mobilisation with an EC50 of 7 nM. D4 receptor activation had no effect upon either basal or hormone-induced [Ca2+]i transients. 5. These results demonstrate that GH4C1 pituitary cells have at least two types of dihydropyridine sensitive high-threshold calcium currents and that like D2 receptors, human D4 receptors can also regulate calcium channel function.

The series of molecular signals generated as a consequence of a dopamine receptor binding to its physiological ligand, where the pathway proceeds with activation of phospholipase C (PLC) and a subsequent release of inositol trisphosphate (IP3) and diacylglycerol (DAG).

We demonstrate a heteromeric D1-D2 dopamine receptor signaling complex in brain that is coupled to Gq/11 and requires agonist binding to both receptors for G protein activation and intracellular calcium release. The D1 agonist SKF83959 was identified as a specific agonist for the heteromer that activated Gq/11 by functioning as a full agonist for the D1 receptor and a high-affinity partial agonist for a pertussis toxin-resistant D2 receptor within the complex. We provide evidence that the D1-D2 signaling complex can be more readily detected in mice that are 8 months in age compared with animals that are 3 months old, suggesting that calcium signaling through the D1-D2 dopamine receptor complex is relevant for function in the postadolescent brain. Activation of Gq/11 through the heteromer increases levels of calcium/calmodulin-dependent protein kinase IIalpha in the nucleus accumbens, unlike activation of Gs/olf-coupled D1 receptors, indicating a mechanism by which D1-D2 dopamine receptor complexes may contribute to synaptic plasticity.

Cytokinesis, the last step during cell division, is a highly coordinated process that involves the relay of signals from both the outside and inside of the cell. We have a basic understanding of how cells regulate internal events, but how cells respond to extracellular cues is less explored. In a systematic RNAi screen of G protein-coupled receptors (GPCRs) and their effectors, we found that some GPCRs are involved in cytokinesis. RNAi knockdown of these GPCRs caused increased binucleated cell formation, and live cell imaging showed that most formed midbodies but failed at the abscission stage. OR2A4 (olfactory receptor, family 2, subfamily A, member 4) localized to cytokinetic structures in cells and its knockdown caused cytokinesis failure at an earlier stage, likely due to effects on the actin cytoskeleton. Identifying the downstream components that transmit GPCR signals during cytokinesis will be the next step and we show that GIPC1 (GIPC PDZ domain containing family, member 1), an adaptor protein for GPCRs, may play a part. RNAi knockdown of GIPC1 significantly increased binucleated cell formation. Understanding the molecular details of GPCRs and their interaction proteins in cytokinesis regulation will give us important clues about GPCRs signaling as well as how cells communicate with their environment during division.

Any process that increases the concentration of calcium ions in the cytosol that occurs as part of a PLC-activating G-protein coupled receptor signaling pathway. G-protein-activated PLC hydrolyses phosphatidylinositol-bisphosphate (PIP2) to release diacylglycerol (DAG) and inositol trisphosphate (IP3). IP3 then binds to calcium release channels in the endoplasmic reticulum (ER) to trigger calcium ion release into the cytosol.

The human dopamine D2L (long form) and D2S (short form) receptors were expressed separately in mouse Ltk- fibroblast cells to investigate whether there is a difference in transmembrane signaling of these D2 receptors. Both receptors induced two signals, a phosphatidylinositol-linked mobilization of intracellular calcium and an inhibition of cyclic adenosine 3'-5' monophosphate (cAMP) accumulation, each with similar response magnitudes and identical pharmacology. Both calcium and cAMP signals were sensitive to pretreatment with pertussis toxin (PTX), indicating mediation by coupling to Gi/Go proteins. However, the two forms of D2 receptor were distinguished by acute prior activation of protein kinase C (PKC) with 12-O-tetradecanoyl 4 beta-phorbol 13-acetate (TPA): TPA blocked the D2S-mediated increase in cytosolic free calcium concentration ([Ca2+]i) in a concentration-dependent manner (between 10 nM and 1 microM), whereas the D2L receptor-induced increase in [Ca2+]i was resistant to TPA and was only partially (60%) inhibited by 100 microM TPA. By contrast, TPA did not alter the inhibition of cAMP accumulation induced by activation of either D2S or D2L receptors. We conclude that, in the L cell system, prior activation of PKC differentially modulates the transmembrane signaling of the D2L and D2S receptors, preferentially inhibiting the D2S receptor-mediated calcium signal but not altering the dopamine-induced inhibitory cAMP signal of either receptor subtype.

Despite a high degree of sequence homology, the dopamine D2 and D3 receptors have substantially different second messenger coupling properties. We have used chimeric D2/D3 receptors to investigate the contribution of the intracellular loops to the signaling properties of these receptors. In HEK 293 cells, D2 receptors inhibit prostaglandin E1-stimulated cyclic AMP levels by >90%, whereas D3 receptors inhibit cyclic AMP accumulation by only 20%. In chimeras that have the second or third intracellular loop, or both loops simultaneously, switched between the D2 and D3 receptors, the maximal inhibition of adenylyl cyclase is 60-90%. In addition, the potency of quinpirole to inhibit adenylyl cyclase activity at some of the chimeras is altered compared with the wild-type receptors. It appears that the intracellular loops of the D3 receptor are capable of interacting with G proteins, as when these loops are expressed in the D2 receptor, the chimeras inhibit adenylyl cyclase similarly to the wild-type D2 receptor. Our data suggest that the overall conformation of the D3 receptor may be such that it interacts with G proteins only weakly, but when the intracellular loops are expressed in another context or the D3 receptor structure is altered by the introduction of D2 receptor sequence, this constraint may be lifted.

Binding of dopamine receptor ligands to human D2 and D3 receptors was characterized in Chinese hamster ovary (CHO) cells using the dopamine D2 receptor antagonist [125I] iodosulpiride. Only limited binding selectivity was observed for known dopamine D2 receptor antagonists from a variety of chemical classes, which included haloperidol, chlorpromazine, sulpiride, pimozide and cis flupenthixol. The most selective compound from this group were (+)butaclamol and domperidone which showed 5-fold D3 selectivity. A number of high affinity dopamine receptor agonists, including apomorphine and bromocriptine, also failed to demonstrate selectivity. In contrast, the natural ligand dopamine and the efficacious synthetic agonists quinpirole, (+)4-propyl-9-hydroxynapthoxazine (PHNO), 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (6,7-ADTN), 7-OH DPAT and N-0434 showed marked apparent human dopamine D3 (hD3) receptor selectivity. In the aminotetralin series, this selectivity was observed preferentially with analogs of the 6,7-rotamer compared with compounds from the 5,6-rotamer series. Functional coupling of the hD3 receptor was investigated in a number of cell lines in which the hD3 receptor was stably expressed, including CHO cells, the neuroblastoma-glioma hybrid cell line NG108-15 and a rat 1 fibroblast cell line. There was no evidence of functional coupling of the hD3 receptor to adenylate cyclase, arachidonic acid release, phospholipase C activation, K+ currents or calcium mobilization in any of the cell lines examined. Furthermore, guanine nucleotides failed to inhibit the binding of [3H] N-0437 to hD3 receptors in any of the three cell lines. There may be a number of explanations for these results. These cell lines may not have the appropriate G-protein or secondary messenger systems that are coupled to the hD3 receptor in situ. Alternatively, this receptor may couple by a mechanism that is as yet undefined. The finding that a wide range of structurally diverse human dopamine D2 (hD2) receptor agonists have an apparent hD3 selectivity may imply that the hD3 receptor exists predominantly in a high affinity state.

Any process that modulates the frequency, rate, or extent of the self-propelled movement of a cell or organism from one location to another in a behavioral context; the aspect of locomotory behavior having to do with movement.

Any process that modulates the frequency, rate or extent of the directed movement of potassium ions (K+) into, out of or within a cell, or between cells, by means of some agent such as a transporter or pore.

Any process that modulates the frequency, rate or extent of the directed movement of sodium ions (Na+) into, out of or within a cell, or between cells, by means of some agent such as a transporter or pore.

Any process that modulates the frequency, rate or extent of synapse structural plasticity. Synapse structural plasticity is a type of cytoskeletal remodeling; this remodeling is induced by stimuli that can lead to long term potentiation and it can be activity-dependent or -independent. Examples of cytoskeletal changes include the formation of new spines and increase in spine size; this can be accompanied by the insertion of greater numbers of glutamate (or other neurotransmitter) receptors into the post-synaptic membrane.

Any process that modulates the frequency, rate or extent of GABAergic synaptic transmission, the process of communication from a neuron to another neuron across a synapse using the neurotransmitter gamma-aminobutyric acid (GABA).

Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of an amphetamine stimulus. Amphetamines consist of a group of compounds related to alpha-methylphenethylamine.

Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a cocaine stimulus. Cocaine is a crystalline alkaloid obtained from the leaves of the coca plant.

Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a drug stimulus. A drug is a substance used in the diagnosis, treatment or prevention of a disease.

Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a histamine stimulus. Histamine, the biogenic amine 2-(1H-imidazol-4-yl)ethanamine, is involved in local immune responses as well as regulating physiological function in the gut and acting as a neurotransmitter.

OBJECTIVE: von Willebrand factor (VWF) is acutely released from endothelial cells in response to numerous calcium-raising agents (e.g. thrombin, histamine) and cAMP-raising agents (e.g. epinephrine, adenosine, vasopressin). In contrast, very few inhibitors of endothelial VWF secretion have been described. The neurotransmitter dopamine is a modulator of exocytosis in several endocrine cells, and is possibly involved in the regulation of several endothelial cell functions. We therefore investigated the effect of dopamine on endothelial VWF secretion. RESULTS: Dopamine, D2/D3- and D4-specific agonists inhibited histamine- but not thrombin-induced VWF secretion. Expression of dopamine D2, D3 and D4 receptors was demonstrated by reverse transcription polymerase chain reaction (RT-PCR) in both human aortic (HAEC) and umbilical vein (HUVEC) endothelial cells. D2-D4 agonists did not inhibit histamine-induced rise in [Ca(2+)](i): they inhibited histamine-induced secretion even in the absence of extracellular calcium. Thus, the dopamine effects are not mediated by [Ca(2+)](i)-dependent signalling. D2/D3- and D4-specific agonists inhibited neither the rise in cAMP nor VWF secretion in response to epinephrine and adenosine, arguing against an effect on cAMP-mediated signalling. D1 and D5 receptors were not detected in HAEC or HUVEC by RT-PCR, and the D1/D5-specific agonist SKF 38 393 failed to modulate VWF secretion, arguing against a role for these receptors in endothelial exocytosis. CONCLUSIONS: Dopamine inhibits histamine-induced endothelial exocytosis by activating D2-D4 receptor, via a mechanism distinct from [Ca(2+)](i)-or cAMP-mediated signaling. In contrast, D1 and D5 receptors are not functionally expressed in cultured endothelial cells. Dopamine agonists may be useful as inhibitors of endothelial activation in inflammation and cardiovascular disease.

Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a stimulus indicating lowered oxygen tension. Hypoxia, defined as a decline in O2 levels below normoxic levels of 20.8 - 20.95%, results in metabolic adaptation at both the cellular and organismal level.

Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a light stimulus, electromagnetic radiation of wavelengths classified as infrared, visible or ultraviolet light.

Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a morphine stimulus. Morphine is an opioid alkaloid, isolated from opium, with a complex ring structure.

The series of events required for an organism to receive an olfactory stimulus, convert it to a molecular signal, and recognize and characterize the signal. Olfaction involves the detection of chemical composition of an organism's ambient medium by chemoreceptors. This is a neurological process.

Keywords

Receptors which transduce extracellular signals across the cell membrane. At the external side they receive a ligand (a photon in case of opsins), and at the cytosolic side they activate a guanine nucleotide-binding (G) protein. These receptors are hydrophobic proteins that cross the membrane seven times.

Protein which is part of a reference proteome. Reference proteomes are a subset of proteomes that have been selected either manually or algorithmically according to a number of criteria to provide a broad coverage of the tree of life and a representative cross-section of the taxonomic diversity found within UniProtKB, as well as the proteomes of well-studied model organisms and other species of interest for biomedical research.